Various techniques exist to implement microscope-based imaging of biological objects, including cells and collections of cells, such as tissue. A traditional approach utilizes conventional light microscopy combined with one or more light filters and computer software programs for analyzing the image acquired from the microscope. The wavelengths of the light filters can be matched to the colors of the stain and counterstain. The filters allow a technician to identify, classify and then measure differences in the optical density of specific colors of light transmitted through stained portions of tissue sections. Some more advanced imaging systems may perform limited automated recognition of features. The feature recognition further may be combined with automated calculation of feature areas, automated calibration, and automatic calculation of average and integrated optical density.
These and other techniques of microscope-based imaging are widely used in a variety of research and diagnostic applications. One particular application relates to analysis of progenitor cells, which are cells capable of differentiating into one or more different cell types. As an example, one type of progenitor cell is a connective tissue progenitor (CTP) cell. CTP cells are defined as cells that are resident in tissue that can contribute to new tissue formation by giving rise to daughter cells that are capable of differentiating into one or more connective tissue phenotypes (e.g., bone, cartilage, muscle, fibrous tissue, adipose tissue). CTPs include both relatively committed progenitor cells and also a smaller population of upstream multipotent stem cells.
Traditionally, assays of CTPs from bone marrow and other tissues have been performed using colony-forming assays. In these assays, CTPs are plated in-vitro, allowed to attach to a substrate, and detected based upon the appearance of a colony of progeny that form around the original site of attachment. The number of colonies is used as a measure of the colony forming units (CFUs) in the sample. This data in turn can be interpreted to provide information about the concentration and prevalence of CTPs in the original tissue that are activated to proliferate or differentiate under specified experimental conditions.
The number of colonies in a cell culture is typically manually counted, which can be a highly subjective and time consuming process. Automated methods for counting colonies have been developed for bone marrow and hematopoietic colonies; however, detailed information describing colony or cell morphology has not been characterized using any automated image analysis method. The only approach currently available for assessing the performance of the progeny is to employ subcloning strategies. This type of analysis is time consuming, costly, and labor intensive. Furthermore, the cloning is inevitably associated with selective pressures that are likely to alter the characteristics of the subcloned population from that in the original sample.